T cells are necessary and sufficient for the induction of acute allograft rejection, while graft-reactive Abs mediate hyperacute allograft rejection and can contribute to acute and chronic allograft rejection. Inhibition of alloreactive B cells and alloantibody production should enhance long-term allograft survival. One effective approach to controlling alloreactive B cells and alloantibody production is through the induction of alloreactive B cell tolerance. We have successfully induced allograft and B cell tolerance in BALB/c mice to C57BL/6 hearts with transient anti-CD40L monotherapy. The overall focus of this proposal is to define the mechanisms that mediate B allograft tolerance, and to develop assays for the diagnosis of B cell tolerance. Specific Aim 1: Defining the fate of alloreactive 3.83 B cells in tolerant and rejecting recipient. We will use the cardiac transplant model of C57BL/6 hearts-into-BALB/c mice, and BCR-knock-in mouse, 3-83 Igi, as a source of alloreactive B cells (specific for Class I MHC antigens H-2Kk/b) to define how alloreactive B cells are controlled in tolerant recipients. Specifically we will test whether clonal deletion, inappropriate homing, inhibition of proliferation or lack of differentiation into plasma cells are the bases for the control of alloantibody production. We will also test whether alloreactive cells that are tolerized by peripheral mechanisms can be phenotypically distinguished from naive or activated alloreactive B cells. Specific Aim 2: Defining the mechanism of peripheral B cell tolerance. Our preliminary data suggest a model of peripheral regulation of alloreactive B cells. We will define the basis of peripheral B cell tolerance by specifically testing (i) whether B cell tolerance is mediated through regulated T cell help or by direct regulation of B cells, (ii) the relative contribution of regulatory T and dendritic cells to the maintenance of B cell tolerance. We will also test whether mechanisms controlling B cell responses differ from the mechanisms regulating Th1 responses and T-cell mediated alloqraft rejection. Specific Aim 3: Defining tolerant alloreactive B cells in wild-type mice. There are many strengths and caveats associated with the use of transgenic BCR-B cells in the study of immune responses, The caveats have prompted questions regarding the relevance of findings made using transgenic animal models to the clinical situation. To address this concern, we have recently developed a new method to track alloreactive B cells in vivo using allo-MHC tetramers. Using this new experimental approach, we will define the behavior of wild-type alloreactive B cells during allograft rejection and tolerance. Completion of the proposed studies should lead to the definition of the basis for don or-reactive B cell tolerance induced by anti-CD40L therapy, and to the definition of the phenotype of tolerant B cells. These insights could lead to new therapeutic strategies for inducing allograft tolerance and novel assays for the diagnosis of B cell tolerance in the clinic.